Project Summary. Although the cytoskeleton has historically been understood as the structural framework of the cell, the proper function of actin is also required for a diverse array of cellular pathways. The collapse of these cellular processes manifests during aging and exposure to a myriad of stresses, which is in part due to the breakdown of the cytoskeleton under these conditions. Interestingly, the breakdown of the cytoskeleton throughout age has been adopted as common knowledge in the field of aging biology, despite the lack of clear and direct evidence. A major contributor to the lack of these essential studies is the lack of tools available for in vivo, live-cell imaging of the actin cytoskeleton in multi-cellular organisms. Early in my postdoctoral career, I developed a system for robust, tissue-specific, live-cell imaging of the cytoskeleton in the muscle, intestine, and hypodermis of C. elegans, utilizing LifeAct fused to a fluorescent molecule. LifeAct-mRuby reliably binds to F-actin, allowing visualization of functional, filamentous actin in the cells it is expressed. Using this system, I performed an exhaustive characterization of the decline of actin cytoskeletal integrity during aging. This work laid the foundation of my currently ongoing work in identification of novel regulators of the actin cytoskeleton. Having set up a system to interrogate cytoskeletal quality, I can now interrogate novel genes in their potential role for actin regulation. Using this and other platforms, I performed a multi-pronged screening approach to identify novel genetic regulators of actin. These studies combined in vivo live cell imaging of actin filaments, synthetic lethality screening with known regulators of the actin cytoskeleton, and both transcriptome analysis and whole genome CRISPR-Cas9 screening of organisms experiencing actin stress. Cross-referencing these rich datasets has revealed two critical nodes of genes: 1) modifiers of chromatin state and their downstream transcriptional regulators and 2) genes involved in lipid storage and global lipid homeostasis. In Aim 1.1, I hypothesize that a general chromatin state exists to promote a healthy transcriptome for proper cytoskeletal form and function, and that this breaks down as a function of age. Moreover, a healthy metabolic state can work either upstream of ? or independent of ? chromatin remodeling to also promote cytoskeletal health. In Aim 1.2, I propose to study whether any of the identified processes can function in a tissue- specific manner and a cell non-autonomous manner, by answering two questions: 1) is overexpression of chromatin remodeling or lipid homeostasis factors in a single tissue sufficient to preserve organismal lifespan? and 2) does overexpression of these genes in neurons drive protection of the actin cytoskeleton in peripheral tissue? Aim 2 uses 2 biochemical approaches to assess cytoskeletal function. First, proximity labeling will be used to characterize novel protein interactors of actin important for proper form and function. Second, we are building a tool for a biochemical approach for quantifying actin function with single cell resolution. This study will open exciting avenues of research in understanding the role of cytoskeletal function on physiological aging.